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1521-009X/46/6/794804$35.00 https://doi.org/10.1124/dmd.117.078709 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 46:794804, June 2018 Copyright ª 2018 by The Author(s) This is an open access article distributed under the CC BY-NC Attribution 4.0 International license. Structural Studies of Multidrug Resistance Protein 1 Using AlmostCysless Template s Daria N. Trofimova and Roger G. Deeley Division of Cancer Biology and Genetics, Queens Cancer Research Institute, Kingston, Ontario, Canada Received September 20, 2017; accepted February 21, 2018 ABSTRACT Multidrug resistance protein, MRP1 (ABCC1) is a broad-spectrum ATP-binding cassette transporter that plays a major role in defense against dietary and environmental toxicants, in addition to contrib- uting toward multidrug resistance of certain types of malignancy. Elucidating the molecular structure of hMRP1 is key to determining its mechanism of substrate recognition and transport. Here, we report the first successful attempt using cysteine-scanning muta- genesis coupled with cross-linking studies to probe the structure of hMRP1 in its native environment of the cell membrane or in membrane vesicles. We have established that an active 3Cys DMRP1 (MRP 2041531 ) mutant, described in previous studies from our laboratory, is a suitable template with which to generate single- and double-cysteine mutants for performing cysteine mutagenesis studies. We have now used 3Cys DMRP1 to probe the arrangement of several TM segments, as well as the location of individual amino acids in these regions. Cysteine residues were introduced into TMs 8, 14, 15, and 16 of 3Cys DMRP1. The mutants were then subjected to chemical cross-linking analyses, and cross-linking was detected between the following cysteine pairs: Cys388 (TM7) and I1193C (TM16); Cys388 (TM7) and E1144C (TM15); R433C (TM8) and E1144C (TM15); and R433C (TM8) and T1082C (TM14). The aqueous acces- sibility of these residues and the possible implications of the differences between the open and closed states of the protein are also discussed. Moreover, using competition experiments involving a well characterized substrate and a cross-linking reagent for probing the Cys388/ I1193C mutant, we have defined these amino acid positions as a component of the potential site for estrone sulfate binding. Introduction Multidrug resistance protein MRP1 (ABCC1) belongs to the superfamily of ATP-binding cassette (ABC) transport proteins. MRP1 is a broad-specificity, transmembrane transporter that can efflux a vast range of amphiphilic organic anions, including many glutathione (GSH), glucuronate, and sulfate conjugates, as well as free GSH (Slot et al., 2011). It is ubiquitously expressed in the human body, with relatively high levels in the lung, testes, kidney, and blood organ barriers (Cole et al., 1992; Bakos and Homolya, 2007). The widespread tissue distribution of MRP1 implies an important physiologic role. However, an essential role in normal physiology remains to be firmly established. Studies in MRP1-null mice have revealed a number of intriguing potential functions, and MRP1 has been proposed to play a protective role in cells that express this protein (Leslie et al., 2005). Clinically, MRP1 is the most extensively studied member of the multidrug resistance protein (MRP) subfamily of ABC transporters, particularly with respect to the development of multidrug resistancereversing agents. Its expression has been strongly linked to chemotherapy response, time to relapse, and/or disease outcome in a number of tumor types, including breast, lung, prostate, and neuroblastoma (Chan et al., 1997; Hipfner et al., 1999). The protein contains 17 transmembrane (TM) helices arranged in three polytopic transmembrane-spanning domains (MSDs), and two cytoplasmic nucleotide-binding domains (NBDs) (Cole, 2014). Bio- chemical and computational analyses indicate that TMs 15 constitute MSD0, and TMs 611 and TMs 1217 constitute MSD1 and MSD2, respectively (Bakos et al., 1996; Hipfner et al., 1997). Elucidating the arrangement of the TM segments, as well as the location of individual amino acids in them, is key to determining the mechanism of MRP1 substrate recognition and transport. TMs form the translocation pore of the protein and are also the site of substrate binding, as established through photoaffinity labeling and extensive mutagenesis studies (Deeley and Cole, 2006; Deeley et al., 2006). The drug-binding site for MRP1 has been proposed to be multipartite in nature, with substrates establishing different molecular contacts within the binding site (Deeley and Cole, 2006). We have used cysteine mutagenesis, combined with cross-linking analysis, to investigate the relative position of residues within the hMRP1 TM helices that are predicted to be near the membrane/cytosol interface. Ideally, this strategy involves the introduction of two or more cysteine residues into an otherwise cysteineless (cysless) variant of a protein of interest. However, human MRP1 contains 25 cysteines and it This work was supported by the Canadian Institutes of Health Research [Grant MOP-97877]. https://doi.org/10.1124/dmd.117.078709. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: aa, amino acid; ABC, ATP-binding cassette; AMP-PNP, adenylyl imidodiphosphate; cryo-EM, cryoelectron microscopy; CuPhen, copper phenanthroline; cysless, cysteineless; GSH, glutathione; HEK, human embryonic kidney; LTC 4 , leukotriene C4; M5M, 1,5-pentanediyl bismethanethiosulfonate; M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate; M17M, 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl bismethanethio- sulfonate; mAb, monoclonal antibody; MRP1, multidrug resistance protein 1; DMRP1, MRP 2041531 ; MSD, transmembrane-spanning domain; NBD, nucleotide-binding domain; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; PM, plasma membrane; PNGase F, peptide:N-glycosidase F; TM, transmembrane. 794 http://dmd.aspetjournals.org/content/suppl/2018/03/23/dmd.117.078709.DC1 Supplemental material to this article can be found at: at ASPET Journals on April 11, 2021 dmd.aspetjournals.org Downloaded from

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1521-009X/46/6/794–804$35.00 https://doi.org/10.1124/dmd.117.078709DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 46:794–804, June 2018Copyright ª 2018 by The Author(s)This is an open access article distributed under the CC BY-NC Attribution 4.0 International license.

Structural Studies of Multidrug Resistance Protein 1 Using “Almost”Cysless Template s

Daria N. Trofimova and Roger G. Deeley

Division of Cancer Biology and Genetics, Queen’s Cancer Research Institute, Kingston, Ontario, Canada

Received September 20, 2017; accepted February 21, 2018

ABSTRACT

Multidrug resistance protein, MRP1 (ABCC1) is a broad-spectrumATP-binding cassette transporter that plays a major role in defenseagainst dietary and environmental toxicants, in addition to contrib-uting toward multidrug resistance of certain types of malignancy.Elucidating the molecular structure of hMRP1 is key to determiningits mechanism of substrate recognition and transport. Here, wereport the first successful attempt using cysteine-scanning muta-genesis coupled with cross-linking studies to probe the structure ofhMRP1 in its native environment of the cell membrane or inmembrane vesicles. We have established that an active 3CysDMRP1 (MRP204–1531) mutant, described in previous studies fromour laboratory, is a suitable template with which to generate single-and double-cysteine mutants for performing cysteine mutagenesisstudies. We have now used 3Cys DMRP1 to probe the arrangement

of several TM segments, as well as the location of individual aminoacids in these regions. Cysteine residues were introduced into TMs8, 14, 15, and 16 of 3CysDMRP1. Themutantswere then subjected tochemical cross-linking analyses, and cross-linking was detectedbetween the following cysteine pairs: Cys388 (TM7) and I1193C(TM16); Cys388 (TM7) and E1144C (TM15); R433C (TM8) and E1144C(TM15); and R433C (TM8) and T1082C (TM14). The aqueous acces-sibility of these residues and the possible implications of thedifferences between the open and closed states of the protein arealso discussed. Moreover, using competition experiments involvinga well characterized substrate and a cross-linking reagent forprobing the Cys388/ I1193C mutant, we have defined these aminoacid positions as a component of the potential site for estronesulfate binding.

Introduction

Multidrug resistance protein MRP1 (ABCC1) belongs to thesuperfamily of ATP-binding cassette (ABC) transport proteins. MRP1is a broad-specificity, transmembrane transporter that can efflux a vastrange of amphiphilic organic anions, includingmany glutathione (GSH),glucuronate, and sulfate conjugates, as well as free GSH (Slot et al.,2011). It is ubiquitously expressed in the human body, with relativelyhigh levels in the lung, testes, kidney, and blood organ barriers (Coleet al., 1992; Bakos and Homolya, 2007). The widespread tissuedistribution of MRP1 implies an important physiologic role. However,an essential role in normal physiology remains to be firmly established.Studies in MRP1-null mice have revealed a number of intriguingpotential functions, and MRP1 has been proposed to play a protectiverole in cells that express this protein (Leslie et al., 2005).Clinically, MRP1 is the most extensively studied member of the

multidrug resistance protein (MRP) subfamily of ABC transporters,particularly with respect to the development of multidrug resistance–reversing agents. Its expression has been strongly linked to chemotherapy

response, time to relapse, and/or disease outcome in a number of tumortypes, including breast, lung, prostate, and neuroblastoma (Chan et al.,1997; Hipfner et al., 1999).The protein contains 17 transmembrane (TM) helices arranged in

three polytopic transmembrane-spanning domains (MSDs), and twocytoplasmic nucleotide-binding domains (NBDs) (Cole, 2014). Bio-chemical and computational analyses indicate that TMs 1–5 constituteMSD0, and TMs 6–11 and TMs 12–17 constitute MSD1 and MSD2,respectively (Bakos et al., 1996; Hipfner et al., 1997).Elucidating the arrangement of the TM segments, as well as the

location of individual amino acids in them, is key to determiningthe mechanism of MRP1 substrate recognition and transport. TMs formthe translocation pore of the protein and are also the site of substratebinding, as established through photoaffinity labeling and extensivemutagenesis studies (Deeley and Cole, 2006; Deeley et al., 2006). Thedrug-binding site for MRP1 has been proposed to be multipartite innature, with substrates establishing different molecular contacts withinthe binding site (Deeley and Cole, 2006).We have used cysteine mutagenesis, combined with cross-linking

analysis, to investigate the relative position of residues within thehMRP1 TM helices that are predicted to be near the membrane/cytosolinterface. Ideally, this strategy involves the introduction of two or morecysteine residues into an otherwise cysteineless (cysless) variant of aprotein of interest. However, human MRP1 contains 25 cysteines and it

This work was supported by the Canadian Institutes of Health Research [GrantMOP-97877].

https://doi.org/10.1124/dmd.117.078709.s This article has supplemental material available at dmd.aspetjournals.org.

ABBREVIATIONS: aa, amino acid; ABC, ATP-binding cassette; AMP-PNP, adenylyl imidodiphosphate; cryo-EM, cryoelectron microscopy; CuPhen,copper phenanthroline; cysless, cysteineless; GSH, glutathione; HEK, human embryonic kidney; LTC4, leukotriene C4; M5M, 1,5-pentanediylbismethanethiosulfonate; M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate; M17M, 3,6,9,12,15-pentaoxaheptadecane-1,17-diyl bismethanethio-sulfonate; mAb, monoclonal antibody; MRP1, multidrug resistance protein 1; DMRP1, MRP204–1531; MSD, transmembrane-spanning domain; NBD,nucleotide-binding domain; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; PM, plasma membrane; PNGase F, peptide:N-glycosidase F; TM,transmembrane.

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has not been possible to produce a cysless form of the protein that willtraffic and function normally in animal cells.We have previously created an“almost cysless”DMRP1 (3CysDMRP1), which retains the functionalitiesof hMRP1 (Qin et al., 2012). This protein excludes MSD0 and containscysteines at position 388 (TM7), as well as at positions 1439 and 1479,which are near the COOH-terminal boundary of NBD2.We first verified that 3Cys DMRP1 is a useful model for performing

cysteine cross-linking studies, notwithstanding the presence of the threeendogenous cysteine residues. Second, we introduced cysteine residuesin the other TM helices and determined their ability to be cross-linked bybifunctional reagents, which allowed us to investigate the spatialrelationships among several TM helices. Finally, we also used thesecysteine residues as sensors to probe the aqueous accessibility ofresidues in the putative MRP1 translocation pore, and to investigate thelocalization of substrate binding sites by competition between substratesand cross-linking reagents.We have compared these results obtained with the protein in its native

membrane environment: with an extensively used computer-generatedmodel of hMRP1, whose basis is the crystal structure of the bacterial half-transporter Staphylococcus aureus Sav1866, in the ADP-vanadate-trapped state, which is predicted to correspond to the low-affinity (closed)conformation of the protein (Dawson and Locher, 2006; DeGorter et al.,2008); and with the very recently published structures of bovine MRP1(bMRP1) determined by cryoelectron microscopy (cryo-EM), in bothapo- and substrate-bound forms (Johnson and Chen, 2017).

Materials and Methods

Reagents

1,5-Pentanediyl bismethanethiosulfonate (M5M), 3,6,9,12,15-pentaoxa-heptadecane-1,17-diyl bismethanethiosulfonate (M17M), and 3,6-dioxaoc-tane-1,8-diyl bismethanethiosulfonate (M8M) were purchased from TorontoResearch Chemicals Inc. (Toronto, ON, Canada). Rat monoclonal antibody(mAb) MRPr1 was purchased from Enzo Life Sciences, Inc. (Lausen,Switzerland). Goat Anti-Rat Alexa Fluor 488, fluorescent dye Hoechst33342, Lipofectamine 2000, Dulbecco’s modified Eagle’s medium, and fetalbovine serum were purchased from Invitrogen Canada Inc. (Burlington, ON,Canada). Mouse anti-His tag antibody, E217bG, estrone sulfate, ATP, andadenylyl imidodiphosphate (AMP-PNP) were all purchased from Sigma-Aldrich (St. Louis, MO). Horseradish peroxidase-labeled secondary antibodyand G418 were purchased from Thermo Fisher Scientific (Mississauga, ON,Canada). Unlabeled leukotriene C4 (LTC4) was obtained from Calbiochem (LaJolla, CA), and radiolabeled [14,15,19,20-3H]LTC4 (172 Ci/mmol) waspurchased from Perkin Elmer Life Sciences (Woodbridge, ON, Canada). Allprimers used in this study were synthesized by Integrated DNA Technologies,Inc. (Coralville, IA).

Construction of Mutants by Site-Directed Mutagenesis

Mutations in TM8. Cysteine residues were introduced into 3Cys DMRP1cDNA using QuikChange II Site-Directed Mutagenesis Kit (Stratagene Califor-nia/Agilent Technologies, La Jolla, CA). The template used formutagenesis was apBlueScript II KS(+) vector containing a BamHI/Bsu36I fragment of humanMRP1 cDNA [corresponding to 283–616 amino acids (aa)] containing C375I,C555A, and C563A mutations. A list of primers can be found in supplementalmaterials (Supplemental Table SI 1). After successful replacement and verifica-tion of the mutation by sequencing, which was performed at the Centre forApplied Genomics (Toronto, ON, Canada), the BamHI/Bsu36I fragment wascloned back into C-term 9xHis-tagged 3Cys DMRP1 cDNA in pcDNA 3.1(–)vector for the expression of mutant proteins in human embryonic kidney(HEK293) cells.

Mutations in TMs 14, 15, and 16. The pBlueScript II KS (+) vector with aBsu36I/EcoRI fragment of humanMRP1 cDNA (corresponding to 617–1294 aa),containing C682A, C730A, C744A, C984A, C1047A, C1105A, C1205A, andC1209A mutations, was used as the template for mutagenesis. A Bsu36I/EcoRI

fragment was cloned into C-term 9xHis-tagged 3CysDMRP1 cDNA in a pcDNA3.1(–) vector. In the case of mutants R433C/E1144C, R433C/S1145C,R433C/D1081C, and R433C/T1082C, a Bsu36I/EcoRI fragment was insertedinto R433C 3Cys DMRP1 9xHis-tagged cDNA in a pcDNA 3.1(–) vector.

Transfection, Selection, and Expression of MRP1 Protein

HEK293 cells at 90% confluency (1 � 106 cells per well) were transfectedwith 4 mg of mutant cDNA using Lipofectamine 2000, according to themanufacturer’s instructions. After 6 hours of incubation at 37�C, transfectionmedia was replaced by fresh Dulbecco’s modified Eagle’s medium supple-mented with 7.5% fetal bovine serum. Four rounds of selection with 700 mg/mlG418 were performed. Cells were transferred and expanded on 150-mmdiameter plates until confluency. Cells were then incubated at 28�C for another24 hours in the presence of 50 mM sodium butyrate and absence of G418.Confluent cells were pelleted, then washed in TB (Tris 50 mM, sucrose250 mM, pH 7.4) supplemented with 1.25 mM CaCl2 and protease inhibitorcocktail (Hoffmann-La Roche Limited, Mississauga, ON, Canada) before beingfrozen at 280�C until further use.

Plasma membrane vesicles of HEK293 were prepared as described previously(Loe et al., 1996). In brief, frozen pellets from HEK293 cells were disrupted bynitrogen cavitation, and plasma membrane vesicles were collected after repeatedcentrifugations and layering on a 35% (w/v) sucrose cushion. Vesicles were thenresuspended in TB through a 27.5-gauge needle, aliquoted, and stored at280�C.Total protein concentration was determined by Bradford assay. Expression levelof 3Cys DMRP1 proteins was determined by immunoblot analysis using anMRP1-specific mAb, MRP1r1 (Bakos et al., 1996). To distinguish betweenendogenously expressed full-length MRP1 and transiently expressed mutants, weused an anti-His tag antibody to probe the immunoblots.

Plasma Membrane Trafficking and Glycosylation Status of Mutant Proteins

Confocal microscopy was performed to verify the localization of mutantswithin cultured cells. In brief, HEK293 cells were seeded on poly-L-lysine-coatedglass coverslips at 1 � 106 cells/well and transfected 24 hours later withLipofectamine 2000 and 4 mg of pcDNA3.1-containing mutants. After 6 hours ofincubation at 37�C, media was replaced with Dulbecco’s modified Eagle’smedium containing 50 mM sodium butyrate and incubated at 28�C for a further16 hours. Cells on coverslips were washed with phosphate-buffered saline (PBS),pH 7.4, then fixed with 95% cold (270�C) ethanol for 10 minutes. 3Cys DMRP1proteins were detected using MRPr1 mAb as described above (Westlake et al.,2003), coupled with Alexa 488 fluorescent anti-rat secondary antibody. Nucleiwere stained with Hoechst 33342 (1:5000). Cells were examined under a QuorumWaveFX-X1 spinning disk confocal microscope (Quorum Technologies Inc.,Guelph, ON, Canada).

To assess the degree of glycosylation, all MRP1 mutants were subjected topeptide:N-glycosidase F (PNGase F) treatment. Membrane vesicles containing10 mg of total protein of 3Cys DMRP1 mutants were treated with 1 ml PNGase F(500 IU; New England Biolabs Ltd., Whitby, ON, Canada) according tomanufacturer instructions. After treatment, protein samples were resolved by7.5% SDS-PAGE and probed with an anti-His tag antibody.

Disulfide Cross-Linking Reaction

Plasma membrane vesicles of mutants 3Cys DMRP1 (total protein: 10 mg)were resuspended in a total volume of 18 ml hypotonic phosphate buffer (10 mMNa2HPO4, 150 mM NaCl, pH 7.2) to open up membrane vesicles and encourageplasma membranes to adopt a sheet-like conformation. The use of hypotonicbuffer ensures that the membrane vesicles are burst open and, thus, that the MRP1protein is accessible from both sides of the membrane (Rothnie et al., 2006). Thisallowed thiosulfonate cross-linking reagents to access cysteines located on bothsides of the cell membrane. The plasma membrane sheet preparation was treatedwith 2 ml of cross-linking reagent (final concentration 0.5 mM) for 15 minutes onice. The reaction was stopped by addition of EDTA solution to a finalconcentration of 80 mM, followed by the addition of 2� SDS sample loadingbuffer [Tris 0.5 M, pH 6.8, 20% (v/v) glycerol, 4.4% (w/v) SDS, and no reducingagent]. Reaction mixtures were then loaded and run on a 7.5% SDS-PAGE gel.Western blotting was used to analyze protein samples using mouse anti-His tagantibody, which recognizes His-tagged mutants of MRP1.

Cross-Linking Studies of DMRP1 795

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The disulfide cross-linking reaction for eachmutant was performed at least twotimes. For each protein mutant two independent expressions and membranevesicle preparations were obtained.

Competition of Substrate with Cross-Linker

For the competition experiments, plasmamembranes of 3CysDMRP1mutantswere first incubated with various concentrations of either LTC4 (0–1000 nM) orestrone sulfate (0–75 mM), in presence of 10 mMMgCl2, for 15 minutes at 22�C.In the case of estrone sulfate, 1 mM S-methyl-GSH was also added. Reactionmixtures were cooled on ice for 5 minutes and cross-linked under the conditionsdescribed above. Samples were then analyzed by immunoblotting with anti-Histag primary antibody.

Aqueous Medium Accessibility Test

For labeling of whole cells, cells transfected with cDNA of a mutant from two150-mm confluent plates were harvested by centrifugation at 230g for 10 minutesat 4�C. Cell pellets were washed twice with 10 ml of cold PBS, then resuspendedwith 5 ml of 2.5 mM 2-aminoethyl methanesulfonate (MTSEA), 5 ml of 10 mMsodium 2-sulfonatoethylmethane thiosulfonate (MTSES), or 5 ml of PBSsolution, and incubated at room temperature for 15 minutes. Different concen-trations of these two compounds were used owing to their specific reactivity withcysteine (Stauffer and Karlin, 1994). The cells were then washed three times with10 ml of cold PBS to remove unreacted chemicals. Cell pellets were frozen andkept at280�C before being used to prepare plasma membrane vesicles accordingto the protocol described above.

To test the effect of MTSEA and MTSES on cross-linking, the plasmamembranes (10 mg of total protein) were resuspended in hypotonic phosphatebuffer and the membrane sheets obtained were treated with 50 mM thiosulfonatecross-linker (M5MorM8M) for 15minutes on ice. The reactions were stopped byaddition of a solution of EDTA containing 2� SDS sample loading buffer, asdescribed above, and analyzed by immunoblot assay.

For labeling of membrane vesicles, untreated mutant cells were harvested bycentrifugation at 230g for 10 minutes at 4�C. Cell pellets were frozen and keptat 280�C. Plasma membrane vesicles were obtained according to the protocoldescribed above. Plasmamembranes (10mg of total proteins) were resuspended inTB and treated with 2.5 mM MTSEA, 10 mM sodium MTSES, or TB for15minutes at room temperature. MTSEAorMTSES reagents were removed frommembrane vesicles using G-50 spin columns equilibrated with TB. Flow-throughfractions rich in membrane vesicles were then treated with 50mMM5M orM8M,or without a cross-linker, for 15 minutes on ice. The reactions were stopped with2� SDS sample loading buffer containing EDTA and no reducing agent, and thenrun on 7.5% SDS-PAGE gel, followed by immunoblot analysis with anti-His tagantibody.

ATP-Dependent Uptake of 3H-Labeled LTC4 by 3Cys DMRP1 and ItsMutants

Vesicular transport activity of 3Cys DMRP1 mutants was measured usingplasma membrane vesicles prepared from transfected HEK293 cells. A one–time-point [3H]LTC4 (50 nM, 20 nCi/reaction) uptake assay was performed at 25�C for3minutes in 50-ml reaction volume containing 4mMATP (or 4mMAMP-PNP inthe control group), 10 mMMgCl2, and membrane vesicles containing 0.1–0.5 mgof MRP1 (estimated by immunoblotting analysis). The reactions were stoppedafter 3 minutes by rapid dilution with 1 ml of ice-cold TB buffer (pH 7.4) andfiltrated through glass fiber filters (type A/E, pore size 1 mm; Pall Life Sciences,Ann Arbor, MI). Filters were washed twice with 4 ml of TB and remainingradioactivity was measured by liquid scintillation counting. ATP-dependent druguptake was determined by subtracting the uptake of LTC4 in the presence ofAMP-PNP from the uptake in the presence of ATP. All transport assays wereperformed in triplicate, with the results presented as mean 6 S.D.

Results

3Cys DMRP1 Is a Suitable Template for Cysteine Mutagenesisand Cross-Linking Studies. We have previously described functionalstudies of the 3CysDMRP1C-term 9xHis tag construct that was used forthe cysteine mutagenesis (Qin et al., 2012). 3Cys DMRP1 is a truncated

form of MRP1 lacking MSD0 (aa 1–203), with all remaining cysteinesreplaced with either Ala, Ser, or Ile, except three endogenous cysteines atpositions 388, 1439, and 1479. These remaining cysteines, located inTM7 (Cys388) and NBD2 (Cys1439 and Cys1479), are depicted inFig. 1. According to the existing model of hMRP1, the distancesbetween Cys388 and Cys1439 and between Cys388 and Cys1479(51 and 66Å, respectively), could not be cross-linked by the bifunctionalthiosulfonate reagent M17M, which has a spacer arm of 23 Å. On theother hand, the predicted distance between Cys1439 and Cys1479 is17 Å, which hypothetically could be cross-linked by the bifunctionalthiosulfonate reagent M17M. To test this hypothesis, cross-linkingreactions of 3Cys DMRP1 9xHis tag were performed with threebifunctional thiosulfonate reagents of various spacer lengths.These reagents cross-link cysteines depending on the distances

between their a-carbon atoms. The bifunctional thiosulfonate reagentsused in the present study—M5M, M8M, and M17M—cross-linkcysteines where their a-carbons are separated by approximately 9, 13,and 23 Å, respectively. If a cross-linking reaction takes place, it can bedetected by a shift in electrophoretic mobility on SDS-PAGE toward alarger molecular-weight species (Loo and Clarke, 1996). All threethiosulfonate reagents failed to induce intramolecular cross-linking ofany of the three endogenous cysteines found in 3Cys DMRP1 (data notshown). However, products with apparent molecular weights in therange of 350 kDa and higher were observed, which were presumed torepresent intermolecular cross-linked products of 3Cys DMRP1. Theseresults suggested that 3Cys DMRP1 was a suitable template forperforming cysteine mutagenesis studies.Cross-Linking Studies of Residues Predicted to Be Close to the

Membrane/Cytosol Interface. One of the essential endogenouscysteines, Cys388, is predicted to be close to the membrane/cytosolinterface in TM7, on the basis of the hMRP1 Sav1866 model, with itsside chain possibly oriented toward TM16 (DeGorter et al., 2008). Thus,we began our studies by examining the ability of Cys388 to becomecross-linked to Cys residues introduced into TM16 (Fig. 2A). We choseto mutate the I1193 and Y1190 residues, as their side chains arepredicted to point toward TM7; their distances from Cys388 areapproximately 6 and 6.7 Å, respectively. Notably, Y1190 has beenshown to be important for transport activity of MRP1. The substitutionof Y1190with Ala, Ser, or Val reduced the transport of several substrates(Conseil et al., 2005) but did not significantly alter the expression ofMRP1. On the basis of these data we predicted that these sites would begood locations for the introduction of cysteines.cDNAs of all mutants described in this study were transfected into

HEK293 cells, grown until confluency, and then harvested, frozen, andlater used to obtain membrane vesicles. Confocal microscopy showedcorrect mutant trafficking, i.e., the proteins resided mostly in the plasmamembrane (PM). A confocal microscopy image of mutant I1193C 3CysDMRP1 is provided in the supplementary materials section (Supple-mental Fig. SI 1). Immunoblotting experiments indicated that I1193Cand Y1190C mutants were successfully expressed. The level of mutantexpression was approximately 50% lower than that of 3CysDMRP1. Toassess the degree of glycosylation, both mutant proteins were subjectedto PNGase F treatment, which showed the presence of fully glycosylatedproteins (Supplemental Fig. SI 2).Aliquots of membrane vesicles containing 10 mg of total protein were

resuspended in phosphate buffer and treated with thiol cross-linkingreagents [M5M, M8M, M17M, and copper phenanthroline (CuPhen)]for 15 minutes on ice. Immunoblot analysis of mutants separated bySDS-PAGE revealed that a cross-linking reaction occurred in mutantI1193C 3Cys DMRP1, between Cys388 and Cys1193 (Fig. 3A), but notin mutant Y1190C 3Cys DMRP1 (data not shown). The band of cross-linked protein (X-link) had an apparent molecular mass of 220 kDa and

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was lost in the presence of reducing agent, suggesting the presence of aninternal thiosulfonate bridge, as expected (data not shown).The three thiosulfonate cross-linking reagents—M5M (9 Å), M8M

(13Å), andM17M (23Å)—all generated a cross-linked product with theI1193C mutant. In contrast, CuPhen, the shortest of the cross-linkers(5 Å), failed to do so. These results suggested that TMs 7 and 16 aresituated adjacent to each other at themembrane/cytosol interface, greaterthan 5 Å apart, but less than 9 Å. This finding is in agreement with theSav1866 model of hMRP1, where TMs 7 and 16 helices are locatedclose together, with a predicted distance of 6 Å between Cys388 andI1193. However, in recently published cryo-EM molecular structure ofbMRP1 (Johnson and Chen, 2017) which is depicted in its apo state, Cys388 points toward TM8 rather than TM16 (Fig. 2B). In such anorientation cross-linking of Cys388 to I1193 (TM16) would not beexpected. The crystal structure of another ABC transporter, mouseP-glycoprotein (P-gp), is also available (Aller et al., 2009). The structurewas obtained in an open conformation. Cys133 in mP-gp occupies acomparable position to Cys388 in hMRP1 (Fig. 2C) and in the crystalstructure projects into the translocation pore of P-gp toward TM16. Thisorientation of Cys 133 in P-gp correlates well with our observation.To determine the optimum cross-linking conditions, we investigated

the effects of different cross-linker concentrations and reaction times.Experiments were carried out at 4�C to reduce thermal motion of theprotein. Cross-linking of the mutant I1193C occurred rapidly, with themajority of the cross-linked product detected within 2 minutes (Fig. 4B).Plasmamembrane vesicles prepared from cells expressing the I1193C

mutant were treated with various concentrations of M5M cross-linker(0–50 mM) for 15 minutes. The majority of the observed cross-linkingoccurred with M5M concentrations of 5–50 mM M5M (Fig. 4A). Withincreasing M5M concentrations, the amount of intramolecular species(X-link) increased, whereas levels of intermolecular species (350–400 kDa) remained largely unchanged. The protein band attributed tonon-cross-linked protein (150 kDa) disappeared in reactions with 5–50

mM cross-linker, confirming that the vast majority of the protein wasmodified by the cross-linker.According to the hMRP1 model, TMs 7 and 15 are situated on

opposing sides of the translocation pore, yet remain relatively close toeach other (Fig. 2A). To investigate the location of TM15 relative toTM7, we selected two amino acids, E1144 and S1145, which arebelieved to project into the translocation pore and to lie close to Cys388in TM7. On the basis of the hMRP1 model, the estimated distance ofCys388 (TM7) from E1144 and S1145 (TM15) was 13.8 and 18.6 Å,respectively. Conseil et al. (2009) substituted E1144 with Ala and foundthat although the E1144A mutant was well expressed, the transport ofestrone sulfate and E217bG was reduced.We produced two mutants of 3Cys DMRP1: E1144C and S1145C.

Plasma membrane sheets of the mutants were subjected to cross-linkingreactions with each of the four thiol cross-linking reagents. Sampleswere analyzed by SDS-PAGE, followed by immunoblot detection. Asshown in Fig. 3B, the longest reagent M17M (23 Å)—but not CuPhen,M5M, or M8M—cross-linked Cys1144 (TM15) to Cys388 (TM7). Incontrast, Cys1145 failed to cross-link to Cys388 (TM7) with any lengthof cross-linker (data not shown). Despite the prediction that the distancebetween these cysteines is short enough to be cross-linked byM17M, thealignment of the side chains may not permit such cross-linking to occur.Nevertheless, given that cross-linking occurred between Cys388 andE1144C, we can conclude that TMs 7 and 15 comprise regions of thetranslocation pore, with an approximate distance between them of lessthan 23 Å, which is in reasonable agreement with the hMRP1 model.The fact that E1144C (TM15) was strongly cross-linked suggests that inthe open conformation, Cys388 (TM7) may be rotated more toward theinterior of the translocation pore than the model hMRP1 in the closedconformation predicts. This is consistent with the observation that thecomparable residue in P-gp (Fig. 2C) projects into the translocation pore(open conformation). It is interesting to note that both bMRP1 structures(Fig. 2B) depict Cys388 faced toward TM8, but not TM15. Contrary to

Fig. 1. Positions of endogenous Cys388, Cys1439, and Cys1497(represented by black spheres) in (A) cryo-EM structure bMRP1(205–1516 aa, PDB ID: 5uj9) and (B) Sav1866-based hMRP1model (300–1531 aa). The sequence of hMRP1 is 91% identical tobMRP1. According to the sequence amino acid, Cys, in human andbovine proteins located at the position 388, however Cys1439 (hMRP1) is located at position 1438 in bMRP1 protein, aswell as Cys1479 is at 1478, respectively. Cys388 is situated inTM7; Cys1439 and Cys1497 are located in NBD2. Images werecreated using PyMOL software (Delano Scientific LLC, San Carlos,CA) The hMRP1 model was aligned to the bMRP1 structure usingalign tool in PyMOL automatically. (A) and (B) show proteinmolecule from the same view point.

TABLE 1

Summary of cross-linking reaction of single and double mutants of 3Cys DMRP1

Numbers correspond to the thiosulfonate reagent that cross-linked cysteine residues in a given mutant. For instance, “5” corresponds toM5M. Dash (–) indicates no cross-linked product detected with any of the three cross-linkers. Empty cells indicate that the cross-linkingreactions were not performed.

TM8 TM16 TM15 TM14Mutant

R433C Y1190C I1193C E1144C S1145C D1081C T1082C

3Cys DMRP1 — — 5, 8, 17 17 — — —

R433C 3Cys DMRP1 5, 8, 17 8, 17 — 17

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our results, such an orientation of Cys 388 and E1144C would appear tobe unfavorable for cross-linking.Cross-linking analyses of I1193C (TM16) and E1144C (TM15)

mutants strongly suggest that in the open conformation, Cys388 (TM7)rotates more toward the interior of the translocation pore. Therefore, weexplored the possibility of cross-linking Cys388 (TM7) with selectedamino acid residues in TM14.TM14 is predicted to be approximately 21 Å from Cys388 (TM7)

(hMRP1 model) and thus could potentially be cross-linked by thelongest reagent, M17M (23 Å). We investigated this possibility byintroducing cysteines at positions 1081 and 1082 in TM14. According tothe hMRP1model, residues at these positions face both the translocationpore and TM7. The predicted distances between C388 and D1081 andbetween C388 and T1082 are 23.6 and 20.5 Å, respectively. Moreover,we have shown previously that mutation of the residue at positionT1082A does not alter the expression or the transport activity of MRP1(Zhang et al., 2003).Membrane vesicles from the 3Cys DMRP1 D1081C and T1082C

mutants were probed with three cross-linking thiosulfonate reagents ofdiffering lengths. Immunoblot analysis revealed no shift in the mobilityof mutants, indicating that the two mutants were not cross-linked. Theabsence of cross-linking in these mutants could be attributed, in part, todifferences between the open and closed conformations of hMRP1.

Although the current model represents hMRP1 in an ADP-trapped(closed) conformation, MRP1 in isolated membrane vesicles most likelyexists in an open conformation. In the latter conformation, TMs 7 and14 may be situated further apart (30–35 Å), as seen in both the P-gpstructure (Aller et al., 2009) and bMRP1 (Johnson and Chen, 2017),which would prevent their cross-linking by even the longest reagent,M17M (23 Å). Taguchi et al. (1997) reported that the addition of ATP inthe presence of vanadate traps MRP1 in a closed state; thus, one wouldexpect cross-linking to occur in the vanadate-trapped state (on the basisof the hMRP1 model). We tested this premise by performing cross-linking experiments in the presence of ATP and vanadate. No cross-linked proteins were formed under these conditions (data not shown).These results therefore failed to provide evidence that TMs 7 and 14 aresituated 20.5–24 Å from each other, as predicted by the hMRP1 model(closed conformation). It is interesting to note that in the recentlyreported LTC4-bound structure of bMRP1 (closed conformation),residues Cys 388 (TM7) and D1081C or Cys 388 (TM7) and T1082C(TM14) were predicted to be 30 or 27 Å apart and thus should not becross-linked even by the longest cross-linking reagent used in this study,which is consistent with our findings.The mutations of TM15 (E1144C, S1145C) and TM14 (D1081C,

T1082C) described above targeted residues predicted to project into thetranslocation pore of MRP1. The opposite side of the translocation pore

Fig. 2. Location of endogenous and introduced Cys residues. Shown is the a-carbon backbone in ribbon representation, as viewed from the extracellular face of themembrane. Residues are depicted in similar colors in three structures as endogenous C388 in red, and mutated residues I1193 pink, Y1190 orange, E1144 yellow, S1145sand, T1082 green, D1081 light green, and R433 blue in (A) human MRP1 model (closed conformation) (DeGorte et al., 2008), (B) cryo-EM structure bovine MRP1 (openconformation, PDB ID: 5uj9) (Johnson and Chen, 2017). (C) Location of corresponding residues in the mouse P-gp structure (open conformation, PDB ID: 4M1M) (Alleret al., 2009).

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is predicted to include TM8, with the ionic side chains of residues R433and Q432 projecting into the pore (Fig. 2A). We chose to introduce acysteine at position 433, given that R433 is the site of a naturallyoccurring Ser polymorphism that results in decreased LTC4 and estronesulfate transport, as well as increased doxorubicin resistance (Conradet al., 2002). The level of expression of the R433C mutant remainedunaltered. Consequently, we generated double mutants with R433C(TM8), together with one of the previously introduced cysteines in TMs14 or 15, to investigate the position of TM8 relative to TMs 14 and 15.We created two mutants (R433C/D1081C and R433C/T1082C) withcysteine substitutions in both TMs 8 and 14. Two other double mutants(R433C/E1144C and R433C/S1145C) were also generated, in which wereplaced cysteines in TMs 8 and 15. Plasma membrane vesicles fromthese mutants were prepared and exposed to various cross-linkers. Themutant of TMs 8 and 15 (R433C/E1144C) was efficiently cross-linked byall three thiosulfonate reagents: M5M (9 Å), M8M (13 Å), and M17M

(23 Å) (Fig. 3C). The R433C/S1145C mutant was cross-linked bythiosulfonate reagents M8M (13 Å) and M17M (23 Å), but not by M5Mor CuPhen (Fig. 3E). The cross-linking ability of these mutants confirmsthe close proximity between TMs 8 and 15 (approximately 9 Å).According to the hMRP1 model (closed conformation), the predicted

distances between R433C (TM8) and E1144C (TM15) and betweenR433C and S1145C (TM15) are 16.1 and 17.1 Å, respectively. In thebMRP1 structure (open conformation), the distances between the samepairs are 17.1 and 17.4 Å (18.0 and 14.6 Å in closed conformation). Bycomparison, in the P-gp crystal structure, these distances are 17.9 and13.8 Å for both closed and open forms of the protein. However, ourexperiments revealed that these distances are much shorter than reportedin all three structures. This might be explained if R433 is actually rotatedmore toward the translocation pore.Cross-linking analysis of TMs 8 and 14 double mutants showed that

R433C/T1082C can be cross-linked byM17M (23Å; Fig. 3D), whereas,

Fig. 3. Cross-linking of 3Cys DMRP1 mutants containing pairs of cysteines. Membranes were prepared from HEK293 cells that expressed mutants I1193C (A), E1144C(B), R433C/E1144C (C), R433C/T1082C (D), and R433C/S1145C (E) of 3Cys DMRP1. Membrane sheets were treated without (no cross-linking) and with cross-linker(CuPhen, M5M, M8M, M17M) for 15 minutes on ice. Final concentration of cross-linking reagents in the reactions was 50 mM. Reactions were stopped by addition of SDSloading buffer containing EDTA and no thiol-reducing agent, and samples were subsequently subjected to immunoblot analysis. Membranes were probed with anti-His tagantibody. Representative immunoblot from at least two independent cross-linking experiments is shown. For each protein mutant two independent protein expressions andmembrane vesicle preparations were obtained. The position of the cross-linked product is indicated (X-link).

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R433C/D1081C showed only barely detectable cross-linking withM17M (data not shown). Neither mutant was cross-linked with any ofthe shorter cross-linkers, such as CuPhen, M5M, and M8M. Cross-linking analysis of R433C/D1081C and R433C/T1082C suggested thatD1081 and T1082 are located quite distantly from R433 despite the factthat TM8 and TM14 are predicted to be adjacent to each other.According to the hMRP1 model the distance between TM8 andTM14, in particular R433 and D1081, is 11.2 Å; thus, we expected tosee cross-linking byM8M (13 Å) but did not. This discrepancy could beattributed to the differences in conformation of the protein between themodel, which represents the protein in a closed form (ADP-trapped),whereas in membrane vesicles, in the absence of a nucleotide and asubstrate, the protein is expected to be in an open conformation. ThebMRP1 structure (apo form) should likewise represent the protein in theopen conformation. However, the distance of the R433C/D1081C pairand R433C/T1082C in bMRP1 is 25.8 and 26.9 Å, respectively, andshould not be cross-linked even by the longest M17M cross-linker(23 Å). This is inconsistent with our findings, as cross-linking wasobserved for the R433C/T1082C mutant. Our experimentally obtaineddistance (more than 13 but less than 23 Å) is similar to bMRP1 (in thepresence of LTC4), where R433/D1081 and R433/T1082 are situated ata distance of 18.3 and 20.5 Å, accordingly.Given that R433C (TM8) can be cross-linked to E1144C (TM15), and

that Cys388 (TM7) cross-links with I1193C (TM16) and E1144C(TM15), we examined whether R433C (TM8) can cross-link withCys388 (TM7). Immunoblot analysis of plasma membranes preparedfrom cells transfected with the R433C mutant and treated with cross-linking reagents revealed no shift in mobility of the mutant protein (datanot shown), suggesting that Cys388 (TM7) and Cys433 (TM8) failed tocross-link, indicating that the spatial orientation of Cys388 and R433Cresidues may prevent cross-linking from occurring.Because we failed to observe cross-linking between R433C (TM8)

and Cys388 (TM7), we introduced point mutations along TM8. Fifteencysteine single-point mutations were introduced along this transmem-brane region, between amino acids 431 and 446, with the goal ofelucidating whether TMs 7 and 8 were situated in close proximity. Eachmutant contains four cysteines, three endogenous and one introduced.

Mutant proteins were expressed, and their correct trafficking wasverified by confocal microscopy. We characterized the plasma mem-brane vesicles by immunoblotting and observed that all the mutantproteins were expressed to a similar degree.We next investigated the possibility of cross-linking the inserted

cysteines with endogenous Cys388 using cross-linking reagents ofvarying lengths (M5M, M8M, M17M, CuPhen). None of the reagentswere able to cross-link the mutants.Cross-linking analysis of proteins with a mutation in TM8 did not

reveal the existence of any cysteine pairs with Cys388 (TM7), despitethe fact that these cysteines are predicted to be close enough for suchcross-linking to occur on the basis of the bMRP1 structures.Competition between Substrates and a Cross-Linker in

R433C/E1144C and I1193C Mutants of 3Cys DMRP1. To assessthe possibility that the cross-linking observed between mutants I1193Cand R433C/E1144C may have been the result of mutationally inducedchanges in the protein tertiary structure, we compared LTC4 uptake bythesemutants with that of 3CysDMRP1. The plasmamembrane vesiclesof both mutants showed ATP-dependent MRP1-mediated uptake of 3H-labeled LTC4. We normalized the amount of MRP1 through densitom-etry analysis of the immunoblots to compare transport activity betweenthese proteins. As summarized in Supplemental Table 2, the level ofLTC4 uptake for both mutants was comparable to that of 3Cys DMRP1.This suggests that tertiary structure of MRP1 was not altered byintroduction of cysteine residues.Mutational evidence exists that TM8, where the mutation R433C was

introduced, forms part of the drug-binding pocket of MRP1 (Grant et al.,2008). Consequently, we examined the effect of substrate binding oncross-linking of the mutant R433C/E1144C 3Cys DMRP1. Plasmamembrane vesicles were preincubated with MRP1 substrates, LTC4 orestrone sulfate, in the presence of S-methyl-GSH, at 23�C for 15minutes.S-methyl-GSH, rather than GSH (Qian et al., 2001), was chosen tostimulate estrone sulfate binding to avoid a possible GSH reaction withthiosulfonates. After preincubation of the membranes with substrate, thesamples were cooled on ice and treated with the cross-linking reagentM8M for 15 minutes, followed by immunoblot analysis. Cross-linkingwas observed in all experiments, independent of the substrate employed.

Fig. 4. Effect of M5M concentration and reaction time on cross-linking of I1193C 3Cys DMRP1. Membranes were prepared fromHEK293 cells transiently expressing the I1193C mutant of 3CysDMRP1. The membranes sheets were incubated with variousconcentrations (0–50 mM) of M5M for 15 minutes on ice (A), orwith 50 mM M5M for the indicated times (minutes) (B). Thereactions were stopped by addition SDS loading buffer contain-ing EDTA but no reducing agent. Samples were then subjected toimmunoblot analysis. The position of cross-linked protein isindicated (X-link).

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The degree of cross-linking was comparable for all substrate concen-trations tested. Our data suggested that neither LTC4 nor estrone sulfateinfluenced the cross-linking reactions of R433C/E1144C 3Cys DMRP1.We also tested the effect of the same substrates (i.e., LTC4 and estrone

sulfate) on the cross-linking of another mutant, I1193C 3Cys DMRP1,located in TM16. The introduced cysteine residue is situated close toY1189 and Y1190, both of which have been reported to aid in thetransport ofMRP1 substrates. The substitution of these residues with Alaor Ser reduced the transport activity of MRP1 (Conseil et al., 2005). Themost dramatic effect was observed on GSH transport, which wasreduced 10–30% compared with wild-type MRP1 (Conseil et al., 2005).These observations suggested that I1193C might also be located in thedrug-binding pocket. Consequently, we examined whether cross-linkinginvolving I1193C was inhibited in the presence of substrate.Membrane vesicles of I1193C 3Cys DMRP1mutant were treated and

analyzed as described above. Immunoblotting revealed that the intensityof the X-link product was decreased in a concentration-dependentmanner by estrone sulfate but not by LTC4. As shown in Fig. 5 andSupplemental Fig. 3, estrone sulfate can eliminate cross-linking ofI1193C 3Cys DMRP1 at concentration above 2.5 mM. Estrone sulfatetreatment was performed in presence of fixed concentration of S-methylGSH, which suggests that estrone sulfate rather than S-methyl GSH isthe active compound in inhibition of the cross-linking reaction. Thesedata suggest that cross-linking occurs within the estrone sulfate bindingpocket of MRP1, in the space between TMs 7 and 16.Residues C388 (TM7), R433C (TM8), E1144C (TM15), and

I1193C (TM16) Are Accessible to the Extracellular Environment.It is unknown whether the translocation pore in MRP1 is accessible tothe extracellular aqueous medium in the closed conformation. ManyMRP1 substrates are organic anions, which are predominantly charged,hydrophilic molecules. Although hydrophobic compounds can betransported by MRP1, their transport often requires the presence of ahydrophilic GSHmolecule (Rothnie et al., 2008). This strongly suggeststhat when the protein is in a high-affinity state, the substrate and/or GSH-binding site(s) must be accessible from the cytosol and that thetranslocation pore is probably closed to the extracellular space.Conversely, when in a low-affinity state, the reverse might be expectedto occur. We performed a series of studies to test this hypothesis and toidentify which residues in the predicted translocation pathway areaccessible to the aqueous phase from the extracellular space.Two thiol-charged compounds, MTSES and MTSEA, have proven to

be useful probes in evaluating the aqueous accessibility of cysteineresidues in channels and transporters (Karlin and Akabas, 1998).MTSEA, a membrane-permeable reagent, can penetrate the membranebilayer and covalently bind to the cysteines of a wide range of proteins.In contrast, MTSES is a membrane-impermeable reagent that, in intactcells, reacts only with cysteines accessible from the extracellular space.If the translocation pore of MRP1 is accessible from the extracellularspace, theoretically both MTSEA and MTSES may potentially inhibitreagent-mediated cross-linking of cysteine pairs on the cytosolic side ofthe membrane (Loo et al., 2004).We first examined the R433C/E1144C mutant of 3Cys DMRP1 to

determine whether these cysteines are accessible from the extracellularmatrix by both reagents. Intact cells transiently expressing theR433C/E1144C mutant were treated with MTSEA, MTSES, or withneither reagent. After extensive washing with buffer, cells wereharvested and frozen. Plasma membrane vesicles were prepared andthen treated with M5M and M8M. The cross-linking reactions wereperformed in a hypotonic phosphate buffer that has been shown totransform the vesicles into membrane sheets (Rothnie et al., 2006) suchthat all unmodified cysteines would be accessible to thiosulfonate cross-linking reagents.

Immunoblot analysis of theMTSEA- andMTSES-treatedmembranesis shown in Fig. 6A. In the case of membrane-permeable MTSEA-treated membranes, an absence of the cross-linked species indicated thatthe cysteine pair was accessible and modified by the MTSEA reagent.The membrane-impermeable MTSES-treated membranes, on the otherhand, displayed three bands (Fig. 6A). According to densitometryanalysis of the immunoblots, 30% of MRP1 remained unmodified(150 kDa), 50% underwent intermolecular cross-linking (350 kDa)—most likely involving cysteines in NDB2—and 20% appeared as anintramolecular cross-link product (X-link; 220 kDa). These resultssuggested that cross-linking between Cys433 and Cys1144 wasinhibited to some extent by the presence of MTSES, which, in turn,implied that Cys433 (TM8) and/or Cys1144 (TM15) were partiallyaccessible to the external aqueous medium.The same approach was used to study the second available mutant,

I1193C 3Cys DMRP1, where the cysteine residues (C388 and C1193)are located in TMs 7 and 16, respectively. MTSEA completely inhibitedcross-linking reactions, and MTSES partially inhibited the formation ofcross-links (Fig. 6B). Likewise, these results indicate that Cys388 and/orthe amino acid residue at position 1193 is/are partially accessible fromthe extracellular side of a membrane.The cross-linking experiments described above were performed using

intact cells in the presence of either MTSEA or MTSES reagents, whichwere then further cross-linked when in the form of membrane sheets.However, the distribution of MRP1 in intact cells is quite complex.Although the majority of MRP1 translocates to the plasma membrane,some of the protein is expressed in different cellular compartments,especially in endocytotic vesicles (Almquist et al., 1995). MRP1 that isnot expressed in the plasma membrane would be inaccessible to themembrane-impermeable MTSES reagent. Consequently, its cysteineswould remain capable of cross-linking, which might explain theobserved residual cross-linking of MRP1 in MTSES-treated cells.Another possible reason for residual cross-linking of MRP1 inMTSES-treated cells could involve the MRP1 conformation. WithMRP1 in intact cells, where exposure to ATP and glutathione cannot bereadily controlled, the protein is expected to exist in both the open (lowaffinity) and closed (high affinity) conformations.To address these issues, we used membrane vesicles instead of intact

cells. Plasmamembrane vesicles ofmutants R433C/E1144C (TMs 8 and15) were treated with MTSEA andMTSES reagents. The reactions wereterminated by quick removal of the reagents through gel-filtrationcolumns. Membrane vesicles were then transformed into membranesheets, as described in Materials and Methods, and probed for cross-linking under standard conditions (50 mM cross-linking; 15 minutes on

Fig. 5. Competition of estrone sulfate with cross-linker in I1193C 3Cys DMRP1.I1193C mutant membrane sheets were preincubated with various concentrations ofestrone sulfate (0–75 mM), 1 mM S-methyl-GSH, and 10 mM MgCl2 for 15 minutesat 23�C. The samples were then cooled to 4�C and treated with 50 mM M8M. Themixtures were subjected to immunoblot analysis with anti-His tag antibody.Densitometric analysis of the immunoblot can be found at Supplemental Fig. SI 3).

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ice). Immunoblot analysis of the MTSEA/MTSES-treated membranesrevealed the absence of cross-linking events, whereas untreatedmembranes were effectively cross-linked (Fig. 7A). On the basis of thisobservation, we concluded that Cys1144 (TM15) and Cys433 (TM8),under ATP-free conditions, were modified by MTSES.Finally, we probed membrane vesicles of the I1193C 3Cys DMRP1

mutant, containing two cysteines located in TMs 7 and 16, withmembrane-permeable and -impermeable MTS reagents. MTSEA andMTSES blocked cross-linking reactions induced by thiosulfonatereagents (Fig. 7B). These results suggested that MTSES has access toCys388 and/or the amino acid at position 1193 (TM16).

Discussion

Cysteine-scanning mutagenesis coupled with cross-linking studiesprovides a powerful tool for investigating structure and dynamics oftransmembrane proteins in the native environment of a cell membrane(Stauffer and Karlin, 1994). Successful cysteine-scanning mutagen-esis requires a fully functional protein, ideally devoid of all cysteines.However, full-length MRP1 presented a challenge to this techniquebecause the protein contains 25 cysteines. We have previouslydescribed the construction of “almost” cysless DMRP1 (3CysDMRP1) that retains the ability to traffic to the PM and to transportseveral MRP1 substrates at a normal rate (Qin et al., 2012). In thisstudy, we first examined the feasibility of using 3Cys DMRP1 as atemplate for cysteine-scanning mutagenesis and cysteine cross-linking studies. We found no evidence of intramolecular disulfidecross-linking involving any of the remaining three endogenouscysteines in the 3Cys DMRP1 template. Furthermore, all 25 mutantsin which additional cysteines had been introduced into the 3CysDMRP1 template remained functional and trafficked to the PM.

Overall, the results of these studies strongly support the poten-tial utility of 3Cys DMRP1 as a template for cysteine-scanningmutagenesis.The spatial relationships among the transmembrane helices were

investigated by disulfide cross-linking experiments using mutants of the3Cys DMRP1 and cross-linkers of differing lengths. Cross-linking wasobserved between cysteine pairs in TM7/TM16, TM7/TM15,TM8/TM15, and TM8/TM14 (Table 1). Based on the lengths of thecross-linking agents, our results indicated that TM7 was 6–9 and 14–24Å apart from TMs 16 and 15, respectively, and that TM8was 6–9 and13–24 Å apart from TMs 15 and 14 (Fig. 8A). These distances are inreasonable agreement with the hMRP1 model and recently obtainedbMRP1 structures (Johnson and Chen, 2017).In contrast, we found considerable discrepancies between distances

predicted by both the hMRP1 model and the cryo-EM structure ofbMRP1 and those suggested by our cross-linking studies with theR433C/E1144C (TMs 8 and 15) mutant. The distance between theseresidues in the hMRP1 model and the cryo-EM structure of bMRP1 ispredicted to be 17.1 Å. However, we found that the two cysteine residuesin the mutant protein could be cross-linked byM5M, indicating that theyare no more than 9 Å apart. The fact that 433C can be cross-linked to1144C suggests that the side chain of amino acid at position 433 isprojecting toward side chain of amino acid position 1144, and thustoward the translocation pore. Neither the bMRP1 structure nor thehMRP1 model supports this finding. In both predicted structures, R433projects away from the translocation pore. However, in bMRP1 (apo andLTC4-bound structures) the adjacent residue, Q432, does project toward

Fig. 7. Effect of MTSEA and MTSES on cross-linking of 3Cys DMRP1 mutants.Membranes prepared from HEK293 cells expressing mutants R433C/E1144C (A)and I1193C (B) were incubated at 23�C for 15 minutes in the presence of 10 mMMTSES or 2.5 mM MTSEA, or without either reagent (none). MTSEA or MTSESreagents were removed from membranes using G-50 spin columns. Plasmamembrane-rich flow-through fractions were then treated with 50 mM M5M orM8M, or without the cross-linker (no CL) for 15 minutes on ice. The reactions weremixed with SDS loading buffer containing EDTA and no reducing agent, and thenrun on 7.5% SDS-PAGE gel, followed by immunoblot analysis with anti-His tagantibody. The position of the cross-linked products is indicated (X-link).

Fig. 6. Effect of MTSEA and MTSES on cross-linking in whole cells expressing3Cys DMRP1 mutants R433C/E1144C (A) and I1193C (B). Cells were treated with10 mM MTSES, 2.5 mM MTSEA, or buffer (none) for 15 minutes at 23�C. Thetreatment was stopped by dilution with ice-cold PBS and cells were washed withPBS to remove the reagents. Membrane sheets were prepared and treated with50 mM M5M or M8M, or without the cross-linker (no CL) for 15 minutes on ice.Protein mixtures were run on 7.5% SDS-PAGE gel, followed by immunoblotanalysis with anti-His tag antibody. The position of the cross-linked product isindicated (X-link).

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Page 10: Structural Studies of Multidrug Resistance Protein 1 Using ... · cytoplasmic nucleotide-binding domains (NBDs) (Cole, 2014). Bio-chemical and computational analyses indicate that

the translocation pore and the distance between the a-carbon atoms ofQ432 and E1143 in bMRP1 (E1143 occupies the equivalent position toE1144 in hMPR1) is predicted to be 12.4 Å, which is nearer to, but stilllonger than, what was detected experimentally by cross-linking. Thisfinding suggests that there may be some structural differences betweennative MRP1 in the lipid bilayer and the models derived from thecrystallographic structure of Sav1866 and the cryo-EM structures ofbMRP.Under physiologic conditions it is possible that MRP1 is not static and

that transmembrane helices are flexible and dynamic (Tanizaki and Feig,2006). Thus the experimental conditions under which cross-linking wasperformed may allow cross-linking of only a subset of potential states ofMRP1, and we cannot exclude existence of other possible states of theprotein that fail to cross-link. To minimize thermal motion of thetransmembrane domains, we performed cross-linking experiments atthe lowest possible temperature. Nevertheless, we cannot exclude thepossibility that remaining thermal motionmay bring two transmembranehelices close enough for cross-linking to occur in a subset ofconformations that do not necessarily represent the predominant stateof the protein. This might be an explanation for the discrepancy indistances between our results and the cryo-EM structure of bMRP1. Onthe other hand, the lack of cross-linking provides a strong indication thatthe distance between the cysteine pairs exceeds the length of a cross-linker under any conditions.Another instance of inconsistency between the bMRP1 structure, the

hMRP1 model, and our cross-linking results occurred with mutantsI1193C and Y1190C (TM16). I1193C, but not Y1190C, cross-linked toCys388 (TM7). According to the hMRP1 model, the Y1190 residue ispredicted to project directly toward Cys388, whereas the side chain ofI1193 projects away from Cys388 and toward the translocation pore.Thus, the cross-linking results are the reverse of what would beanticipated. The bMRP1 structure depicts Cys388 situated on the sideof TM7 facing TM8 and projecting away from the translocation pore.This orientation was not supported by our cross-linking results.Considering that MRP1 in a living cell is usually exposed to a highlevel of ATP and GSH (one of its substrates/modulators) and embeddedin a lipid bilayer, one might expect that both the hMRP1 model (ADP-vanadate-trapped) and the bMRP1 structures (in the absence andpresence of LTC4, no nucleotide) may not totally represent the state ofthe protein in its natural environment. Therefore, our 3Cys DMRP1template may provide a useful comparative tool for probing theconformations of MRP1 in its native environment.In addition, we attempted to probe the substrate-binding site(s) within

3Cys DMRP1 cysteine mutants by investigating the ability of wellcharacterized MRP1 substrates to inhibit cross-linking. One suchsubstrate, estrone sulfate, effectively abrogated cross-linking ofCys388 (TM7) and I1193C (TM16), which suggests that estrone sulfatemay interact with residues in TM7 and/or TM16 that are close to thecytosolic interface of the translocation pore. This finding agrees withprevious mutagenesis experiments in which the mutation of residues1189 or 1190 in TM16 led to reduced transport of several MRP1substrates (Conseil et al., 2005). However, the high-affinity substrate,LTC4, failed to inhibit cross-linking of these cysteines (Cys388 andI1193C). The variable effects of these two substrates are consistent withthe hypothesis that MRP1 contains multiple and somewhat overlappingbinding domains within a larger, flexible pocket (Deeley et al., 2006).Using the same strategy (competition between a substrate and a cross-

linker), we probed another area of the predicted translocation poresituated between R433C (TM8) and E1144C (TM15). In this case, wewere unable to demonstrate substrate-dependent inhibition of cross-linking by any substrates tested, despite the fact that mutation of theseresidues reduces estrone sulfate transport in the case of E1144A (Conseil

et al., 2009) and decreases LTC4 transport in the case of R433S (Conradet al., 2002). Overall, the results suggest that these residues may notinteract directly with the tested substrates but could be located within ornear the substrate-binding pocket. The recent cryo-EM structure ofbMRP1 in the presence of LTC4 (Johnson and Chen, 2017) may providean explanation for the different response of two tested mutants, i.e., thelack of inhibition of cross-linking by any of the substrates tested in thecase of R433C/E1144C mutant and selective inhibition in the case ofC388/I1193C mutant. As can be seen in the bMRP1 structure (Fig. 8B),E1144 and R433 are located quite far from the LTC4 binding pocket,whereas C388 and I1193 are closer. It might be inferred that the presenceof LTC4 would not interfere with cross-linking of R433C/E1144C pairbut might inhibit C388/I1193C cross-linking. Although we observed noinhibition of the cross-linking of C388 and I1193C by LTC4, cross-linking was strongly inhibited by estrone sulfate. This may be attribut-able to the binding of estrone sulfate within the same pocket but in anorientation such that it binds closer to the C388/I1193C residues. Thisresult supports the previously postulated idea of a GSH-binding–induced conformational change that can unmask the estrone sulfatebinding site (Rothnie et al., 2008).

Fig. 8. Structural features of MRP1. (A) Determining the dimensions of the hMRP1translocation pore employing thiol cross-linking reagents. Shown is the arrangement ofTM helices (on the basis of bMRP1 PDB ID: 5uj9) as viewed from the extracellularface of the membrane. Numbers represent distances (in angstroms) deduced by resultsof cross-linking experiments using varied-length cross-linking reagents. (B) Spatialarrangement of cysteine pairs (C388/I1193C and R433C/E1144C) in bMRP1 structurein presence of LTC4 (PDB ID: 5uja). Shown is the view from the plane perpendicularto the lipid bilayer. Dotted lines represent a cross-link in each mutant.

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Finally, we used the C388/I1193C and E1144C/R433C mutants toinvestigate the aqueous accessibility of the binding pocket as awhole. We hypothesized that when MRP1 is expressed in cellmembranes, the substrate and/or GSH-binding site(s) must beaccessible from the cytosol, and that the translocation pore ispresumably closed to the extracellular space. However, our exper-iments with whole cells expressing R433C/E1144C or I1193Cmutants, as well as with membrane vesicles (representing an ATP-free environment), revealed that residues at positions 388 (TM7),433 (TM8), 1144 (TM15), and 1193 (TM16) are accessible to theextracellular aqueous medium. Interestingly, similar results weredescribed for P-gp (Loo et al., 2004), also indicating that the drug-binding site of P-gp is accessible from the aqueous milieu on theextracellular side of a membrane, despite the dissimilar nature of thesubstrates for P-gp and MRP1. Most the P-gp substrates are believedto diffuse freely through the cell membrane, whereas many MRP1substrates are relatively hydrophilic, and the transport of somesubstrates (both hydrophobic and hydrophilic) depends on or isstimulated by hydrophilic GSH molecules.In summary, our results support the suitability of “almost” cysless

DMRP1 as a template for cysteine-scanning mutagenesis coupled withcross-linking studies. Use of this template has provided and maycontinue to provide significant insight into the complex role of MRP1in the binding and transport of various drugs and chemicals.

Acknowledgments

The authors thankMonika Vasa for excellent technical assistance andMatthewGordon for microscopy assistance. We also thank Dr. Gwenaëlle Conseil forfruitful discussions and Anthony Zara for manuscript preparation.

Authorship ContributionsParticipated in research design: Trofimova, Deeley.Conducted experiments: Trofimova.Performed data analysis: Trofimova, Deeley.Wrote or contributed to the writing of the manuscript: Trofimova, Deeley.

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Address correspondence to: Dr. Roger G. Deeley, Division of Cancer Biology andGenetics, Rm. 300, 10 Stuart St., Queen’s University Cancer Research Institute,Kingston, ON K7L 3N6, Canada. E-mail: [email protected]

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